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Inhibitory and Noninhibitory Monoclonal Antibodies to Human Cytochrome P450 2E1

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Laboratory of Molecular Carcinogenesis, National Cancer Institute, National Institutes of Health, 9000 Rockville Pike, Building 37, Room 3E24, Bethesda, Maryland 20892
Cite this: Chem. Res. Toxicol. 1996, 9, 6, 1023–1030
Publication Date (Web):August 29, 1996
https://doi.org/10.1021/tx9600154
Copyright © 1996 American Chemical Society
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Abstract

A panel of 17 hybridomas producing (MAbs) against human cytochrome P450 2E1 (h2E1) was generated by immunizing mice with baculovirus-expressed h2E1. All 17 hybridoma clones gave positive ELISA or immunoblots with either baculovirus- or vaccinia virus-expressed h2E1. Two of the latter were further developed due to their desirable characteristics. MAb 1-73-18 was found to be a powerful inhibitor of P450 h2E1; however, it did not yield a positive immunoblot. MAb 2-106-12 was found to be noninhibitory but formed a strong positive immunoblot with P450 h2E1. These MAbs to h2E1 were highly specific and did not recognize six other human P450s as tested with ELISA or immunoblot analyses. The MAbs to baculovirus-expressed h2E1 also reacted with h2E1 expressed from a vaccinia virus vector system as well as with microsomal fractions of human and acetone-treated rat liver. MAb 1-73-18 inhibited h2E1 enzyme activity catalyzing the metabolism of phenanthrene by 85%, p-nitroanisole by 90%, 4-methylanisole by 60−80%, toluene by 90%, and chlorzoxazone by 90%. The inhibitory MAb 1-73-18 is uniquely useful for determining the contribution of h2E1 to the metabolism of h2E1 substrates in human liver containing multiple P450s. The quantitatively determined contribution of h2E1 to the metabolism of the above substrates ranged from 25% to 75%. Thus, h2E1 was responsible for the following percentages of the total metabolism in human liver:  p-nitroanisole (35%), phenanthrene (23%), methylanisole to cresol (25%), methylanisole to methoxybenzyl alcohol (12%), toluene (40%), and chlorzoxazone (72%). The MAb 2-106-12 forming a strong immunoblot is useful for determining the amount of h2E1 protein in a tissue. Thus the utility of the inhibitory and immunoblot positive MAbs is complementary and can determine both the contribution of h2E1 to the metabolism of specific substrates and the amount of h2E1 protein in human tissue. The analyses of metabolism with the inhibitory MAb 1-73-18 can be generalized and applicable to all h2E1 substrates.

*

 Address correspondence to this author. Telephone (301) 496-6849; Fax (301) 496-8419.

 Abstract published in Advance ACS Abstracts, August 1, 1996.

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This article is cited by 20 publications.

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  2. Esther F.A. Brandon, Petra C.E. van Kesteren, Jan C.H. van Eijkeren, Marc Tienstra, Patricia Lopez Sanchez, Elisa C.M. Tonk, Aldert H. Piersma, Peter M.J. Bos. Implementation of toxicokinetics in toxicity studies – Toxicokinetics of 4-methylanisole and its metabolites in juvenile and adult rats. Regulatory Toxicology and Pharmacology 2015, 73 (1) , 55-64. https://doi.org/10.1016/j.yrtph.2015.06.018
  3. Kristopher W. Krausz, Harry V. Gelboin. Monoclonal Antibody Analyses of Microsomal Human Drug Metabolism and Multifunctional Cytochrome P450. 2012,,https://doi.org/10.1002/9780470921920.edm063
  4. Steven W. Louie, Magang Shou. Drug-Metabolizing Enzymes, Transporters, and Drug-Drug Interactions. 2011,,, 83-149. https://doi.org/10.1002/9780470929278.ch4
  5. Magang Shou, Anthony Y. H. Lu. Antibodies as a Probe in Cytochrome P450 Research. Drug Metabolism and Disposition 2009, 37 (5) , 925-931. https://doi.org/10.1124/dmd.108.025718
  6. Dharamainder Choudhary, Ingela Jansson, Karim Rezaul, David K. M. Han, Mansoor Sarfarazi, John B. Schenkman. Cyp1b1 Protein in the Mouse Eye during Development: An Immunohistochemical Study. Drug Metabolism and Disposition 2007, 35 (6) , 987-994. https://doi.org/10.1124/dmd.106.014282
  7. Harry V. Gelboin, Kristopher Krausz. Monoclonal Antibodies and Multifunctional Cytochrome P450: Drug Metabolism as Paradigm. The Journal of Clinical Pharmacology 2006, 46 (3) , 353-372. https://doi.org/10.1177/0091270005285200
  8. Connie Cheung, Ai-Ming Yu, Jerrold M. Ward, Kristopher W. Krausz, Taro E. Akiyama, Lionel Feigenbaum, Frank J. Gonzalez. THE CYP2E1 -HUMANIZED TRANSGENIC MOUSE: ROLE OF CYP2E1 IN ACETAMINOPHEN HEPATOTOXICITY. Drug Metabolism and Disposition 2005, 33 (3) , 449-457. https://doi.org/10.1124/dmd.104.002402
  9. Blair U. Bradford, Hiroshi Kono, Fuyumi Isayama, Oksana Kosyk, Michael D. Wheeler, Taro E. Akiyama, Lisa Bleye, Kristopher W. Krausz, Frank J. Gonzalez, Dennis R. Koop, Ivan Rusyn. Cytochrome P450 CYP2E1, but not nicotinamide adenine dinucleotide phosphate oxidase, is required for ethanol-induced oxidative DNA damage in rodent liver. Hepatology 2005, 41 (2) , 336-344. https://doi.org/10.1002/hep.20532
  10. Poh-Sing Ng, Susumu Imaoka, Toyoko Hiroi, Mayuko Osada, Toshio Niwa, Tetsuya Kamataki, Yoshihiko Funae. Production of Inhibitory Polyclonal Antibodies against Cytochrome P450s. Drug Metabolism and Pharmacokinetics 2003, 18 (3) , 163-172. https://doi.org/10.2133/dmpk.18.163
  11. Slobodan Rendic. Summary of information on human CYP enzymes: human P450 metabolism data. Drug Metabolism Reviews 2002, 34 (1-2) , 83-448. https://doi.org/10.1081/DMR-120001392
  12. Jens M. Baron, Daniela Höller, Ruth Schiffer, Silke Frankenberg, Mark Neis, Hans F. Merk, Frank K. Jugert. Expression of Multiple Cytochrome P450 Enzymes and Multidrug Resistance-Associated Transport Proteins in Human Skin Keratinocytes. Journal of Investigative Dermatology 2001, 116 (4) , 541-548. https://doi.org/10.1046/j.1523-1747.2001.01298.x
  13. Magang Shou, Tin Lu, Kristopher W. Krausz, Yang Sai, Tianjian Yang, Kenneth R. Korzekwa, Frank J. Gonzalez, Harry V. Gelboin. Use of inhibitory monoclonal antibodies to assess the contribution of cytochromes P450 to human drug metabolism. European Journal of Pharmacology 2000, 394 (2-3) , 199-209. https://doi.org/10.1016/S0014-2999(00)00079-0
  14. Richard C Zangar, Janet M Benson, Vicki L Burnett, David L Springer. Cytochrome P450 2E1 is the primary enzyme responsible for low-dose carbon tetrachloride metabolism in human liver microsomes. Chemico-Biological Interactions 2000, 125 (3) , 233-243. https://doi.org/10.1016/S0009-2797(00)00149-6
  15. Peter J. Korytko, Fred W. Quimby, Jeffrey G. Scott. Metabolism of phenanthrene by house fly CYP6D1 and dog liver cytochrome P450. Journal of Biochemical and Molecular Toxicology 2000, 14 (1) , 20-25. https://doi.org/10.1002/(SICI)1099-0461(2000)14:1<20::AID-JBT3>3.0.CO;2-D
  16. K. W. Krausz, I. Goldfarb, T. J. Yang, F. J. Gonzalez, H. V. Gelboin. An inhibitory monoclonal antibody to human cytochrome P450 that specifically binds and inhibits P4502C9II, an allelic variant of P4502C9 having a single amino acid change Arg144 Cys. Xenobiotica 2000, 30 (6) , 619-625. https://doi.org/10.1080/004982500406444
  17. Harry V Gelboin, Kristopher W Krausz, Frank J Gonzalez, Tian J Yang. Inhibitory monoclonal antibodies to human cytochrome P450 enzymes: a new avenue for drug discovery. Trends in Pharmacological Sciences 1999, 20 (11) , 432-438. https://doi.org/10.1016/S0165-6147(99)01382-6
  18. Tian J Yang, Kristopher W Krausz, Magang Shou, Shen K Yang, Jeroen T.M Buters, Frank J Gonzalez, Harry V Gelboin. Inhibitory Monoclonal Antibody to Human Cytochrome P450 2B6. Biochemical Pharmacology 1998, 55 (10) , 1633-1640. https://doi.org/10.1016/S0006-2952(98)00018-5
  19. Kristopher W. Krausz, Tian Jian Yang, Frank J. Gonzalez, Magang Shou, Harry V. Gelboin. Inhibitory monoclonal antibodies to human cytochrome P450 2D6. Biochemical Pharmacology 1997, 54 (1) , 15-17. https://doi.org/10.1016/S0006-2952(97)00261-X
  20. Frank J. Gonzalez. Overview of Experimental Approaches for Study of Drug Metabolism and Drug-Drug Interactions. 1997,,, 255-277. https://doi.org/10.1016/S1054-3589(08)60210-0

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